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Boyle, J. T. A., Leonard, N. J., and Occolowitz, J. (1969), Biochemistry 8, 3071. Chen, C.-M., and Hall, R. H. (1969), Phytochemistry 8, 1697. Digby, J., and Skoog, F. (1966), PlantPhysiol. 41, 647. Dravnieks, D. E., Burris, R. H., and Skoog, F. (1969), Plant Physiol. 44,866. Fittler, F., Kline, L. K., and Hall, R. H. (1968), Biochemistry 7,940. Fox, J. E. (1966), Plant Physiol. 41, 75. Fox, J. E., and Chen, C.-M. (1967), J Biol. Chem. 242, 4490. Fox, J. E., and Chen, C.-M. (1968), Biochemistry and Physiology of Plant Growth Substances, Wightman, F.. and Setterfield, G., Ed., Ottawa, Runge Press, p 777. Hall, R. H.,Czonka, L., David, H., and McLennan, B. (1967), Science 156,69. Hecht, S . M., Gupta, A. S., and Leonard, N. J. (1970), Anal. Biochem. (in press). Hecht, S. M., Leonard, N. J., Burrows, W. J., Armstrong, D. J . , Skoog, F., and Occolowitz, J. (1469a), Science 166, 1272. Hecht, S. M., Leonard, N. J., Occolowitz, J., Burrows, W. J., Armstrong, D. J., Skoog, F., Bock, R. M., Gillam, I.,
and Tener, G. M. (1969b), Biochem. Biophys. Res. Commun. 35,205. Kende, H., and Tavares, J. E. (1968), Plant Physiol. 43, 1244. Kirby, K. S. (1965), Biochem. J . 96, 266. Kline, L. K., Fittler, F., and Hall, R. H. (1969), Biochemisrry 8,4361, Linsmaier, E. M., and Skoog, F. (1965), Physiol. Plant 18, 100. Linsmaier, E. M., and Skoog, F. (1966), Planta 72,146. Madison, J. T., Everett, G . A., and Kung, H.-K. (1967), J . Biol. Chem. 242, 1318. Madison, J. T., and Kung, H.-K. (1967), J . Bid. Chem. 242, 1324. McChesney, J. D. (1970), Can. J . Botany 48.2357. Peterkofsky, A. (1968), Biochemistry 7,472. Richmond, A,, Back, A., and Sachs, B. (1970), Planta 90,57. Robins, M. J., Hall, R . H., and Thedford, R. (1967), Biochemistry 6 , 1877. Skoog, F., and Armstrong, D. J. (1970), Annu. Rec. Plant Physiol. 2I, 359. Staehelin, M., Rogg, H., Baguley, B. D., Ginsburg, T., and Wehrli, W. (1968), Nature (London) 219,3163. Zachau, H. G., Dutting, D., and Feldman, H. (1966), Angew. Chem. 78, 392.
Photochemical Reactions of Cytosine Nucleosides in Frozen Aqueous Solution and in Deoxyribonucleic Acid* A. J. Varghese
Irradiation of cytidine in frozen aqueous solution with 254-nm ultraviolet radiation produces two types of dimeric product, Cdl and Cd?. Cdl has been identified as a cyclobutane-type dimer. On heating in acidic, neutral, or alkaline aqueous solutions, Cdi is converted mainly to cytidine, cyclobutane-type uridine dimer, and uridine, respectively. Mild acid hydrolysis of the uridine dimer obtained from Cdl gave cyclobutane-type uracil dimer, the infrared and ultraviolet absorption spectra of which are identical with those of the cis-syn isomer. An aqueous solution of Cdr showed an ultraviolet absorption maximum at 314 nm. Acid hydrolysis of Cdr yielded 6-(4’-pyrimidin-2’one)uracil (PO-U) which has absorbance maxima at 305 nm (E 10,800) in acidic and neutral solutions and at 325 nm ABSTRACT:
A
major fraction of the lethal and mutagenic effects of ultraviolet light on biological systems has been attributed to photochemical transformations of pyrimidine bases in the nucleic acids (McLaren and Shugar, 1964). While considerable progress has been made in recent years in understanding the photochemical reactions of thymine and uracil derivatives, much less is known about cytosine and its derivatives (Burr,
* From the Department of Biology, University of Texas a t Dallas, Box 30365, Dallas, Texas 75230. Receiced December 28, 1970. Supported by U. S . Public Health Service Grants GM 13234, FRO 5646, and GM 16547.
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( E 12,000) in alkaline solutions. Irradiation of deoxycytidine in frozen aqueous solution produced two products having properties similar to those of Cdl and Cds. PO-U was also found to be present in acid hydrolysates of irradiated DNA. The proposed mechanism of formation of PO-U involves a cytosine adduct probably derived from an azetidine intermediate. Irradiation of a mixture of thymidine and cytidine, or thymidine and deoxycytidine produced a number of products from which, after acid hydrolysis, thymine dimer, thymine-uracil dimer, uracil dimer, 6-(4’-pyrimidin-2 ’-one)thymine, and 6-(4’-pyrimidin-2 ’-one)uracil were identified. All these products are also present in acid hydrolysates of irradiated DNA.
1968; Fahr, 1968). It has been shown that irradiation of cytidine and cytidylic acid in aqueous solution with ultraviolet light (A 254 nm) adds water across the C5-Csdouble bond to form the 5,6-dihydro-6-hydroxy derivatives (photohydrates) (Johns et a/., 1965; Miller and Cerutti, 1968). However, attempts to demonstrate the formation of photohydrates in native DNA have not been successful (Setlow and Carrier, 1963). On the other hand, there is substantial evidence to indicate that cyclobutane-type dimerization of cytosine residues occurs in irradiated bacterial DNA and the number of cytosine-containing dimers is comparable to the number of thymine-containing dimers at low doses of ultraviolet
PHOTOCHEMICAL REACTIONS OF CYTOSINE NUCLEOSIDES
light (Setlow and Carrier, 1966). In a recent paper we have shown that irradiation of cytosine nucleosides in frozen aqueous solution produces cyclobutane-type dimeric products (Varghese and Rupert, 1971). From acid hydrolysates of irradiated DNA, cyclobutanetype cis-syn-thymine dimer (I) (see Chart I), thymine-uracil dimer (11), 6-(4'-pyrimidin-2 '-0ne)thymine (111), and 5-thyminyl-5,6-dihydrothymine (IV) have been isolated. Since all these products have also been isolated from frozen-solution irradiation of thymine or thymidine (Varghese, 1970), we studied the photochemical reactions of cytosine nucleosides in frozen aqueous solution. Such an investigation should contribute to an understanding of the photochemical alterations of cytosine residues in DNA.
CHART I
o& HN
n
I
111
Experimental Section Materials Cytidine, deoxycytidine, uridine, uracil, and calf thymus DNA (Sigma, Grade 1) were purchased from Sigma Chemical Corp. Dowex 50W X-12 (H+, 100-200 mesh), Dowex 1X-8 (Cl-, 200-400 mesh), and Bio-Gel P-2 (100-200 mesh) were obtained from Bio-Rad Laboratories. All solvents were of reagent grade. [2-14C]Cytidine was obtained from Schwarz BioResearch, Inc. Methods Irradiation. The method of irradiation with ultraviolet light (254 nm) in frozen aqueous solution has been previously described (Varghese, 1970). For labeled samples, the solution contained 10 pCi/200 ml of [2-I4C]cytidine(33.8 mCi/mmole). In the case of mixtures, the solutions were 1 mM with respect to the individual bases of nucleosides. For DNA irradiation conditions were the same as those used for the isolation of cyclobutane-type cis-syn-thymine dimer from DNA (Varghese and Wang, 1967a). Paper Chromatography. The samples were concentrated and streaked on Whatman No. 3MM paper (46 X 57 cm; about 50 mgisheet) and chromatographed by the descending technique. The following solvent systems were used: (A) sec-butyl alcohol saturated with water and (B) n-butyl alcohol-acetic acid-water (80 :12 :30, v/v). Detection and Isolation of Products. From a chromatogram, strips (1 cm wide) were cut out and eluted with water. The ultraviolet absorption spectrum of each fraction was taken. A spectrum different from that of the starting material indicated the presence of a product. Aliquots of 3 ml from each of these fractions were irradiated for approximately 1 min at a distance of 4 cm from two germicidal lamps and the ultraviolet absorption spectrum was again taken. (If cyclobutanetype dimers were present, the absorbance after irradiation will increase at 270 nm for cytosine and at 260 nm for uracil.) For isolation, strips containing the desired product were cut out and extracted thoroughly with water. The extracts were concentrated and the products purified by rechromatography in the same solvent or by column chromatography. Acid Hydrolysis. Trifluoroacetic acid hydrolysis was carried out in sealed tubes at 165" for 90 min. The hydrolysate was chromatographed on papers as described above. For mild acid hydrolysis, the samples were heated in a boiling water bath for 30 min in 4 N hydrochloric acid. The hydrolysate was evaporated to dryness, redissolved in water, and chromatographed on paper. Column Chromatography. For the purification of cyclobutane-type dimeric products of cytosine nucleosides, an aqueous
V 0
H
H
VI
solution containing about 10-15 mg of the sample was applied to a 4 X 90 cm Bio-Gel P-2 column. The column was eluted with water and 20-ml fractions were collected. PO-U was purified by using a Dowex 50W X-12 (H+, 100-200 mesh) column. The sample was applied in aqueous solution to a 2 x 30 cm column. The column was eluted with water and 10-ml fractions were collected. Cyclobutane-type dimeric uridine was purified by a Dowex 1X-8 (formate, 200400 mesh) column. About 50 mg of the sample was dissolved in 5 ml of 0.1 N ammonium hydroxide and applied to a 2 X 30 cm column. Elution was performed with a linear ammonium formate gradient (Weinblum and Johns, 1965). The mixing chamber contained 1.25 ml of formic acid, 5 ml of ammonium hydroxide, and 1 1. of water, while the reservoir contained 2 ml of formic acid, 7.5 ml of ammonium hydroxide, and 1 1. of water. Fractions of 10 ml each were collected and the ultraviolet absorbance spectrum of each fraction was taken. Cyclobutane-type dimeric products were detected by irradiating aliquots of each fraction at 254 nm and observing the increase in absorbance, as mentioned above. Fractions containing the desired product were combined and evaporated to dryness. Cyclobutane-Type cis-syn-Uracil Dimer ( V ) and 6-(4'-Pyrimidin-2'-one)Uracil (PO-U;' V I ) from Uracil. Uracil solution irradiated in the frozen state was evaporated to dryness on a rotary evaporator at 40-45'. The residue (from 1 g of uracil) was extracted with water until the extract showed no ultraviolet absorption maximum above 250 nm. The residue so obtained upon cyrstallization from hot water amounted to 420 mg. A portion (25 mg) of the residue was dissolved in 20 ml of 0.1 N N H 4 0 H and subjected to Dowex 1 x 4 (formate) column chromatography.Two uracil dimers were detected. The fractions constituting the individual peaks were pooled and conAbbreviation used is : PO-U, 6-(4'-pyrimidin-2'-one)uracil. BIOCHEMISTRY,
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H FIGURE 1 : Ultraviolet absorption spectra of Cdl (A) in aqueous solution; (B) after exposure to 254-nm radiation. FIGURE 2: Ultraviolet absorption spectra of Cdl (A) in aqueous solution at pH 2; (B) after exposure to 254-nm radiation; (C) after heating for 5 rnin at 100".
centrated to crystallization. The cyrstalline products were recrystallized from hot water. The major product (16 mg) appeared in fractions 2 8 4 0 and the minor one (1.5 mg) appeared in fractions 45-55. On the basis of acid stability, photoreversal to uracil and infrared absorption spectra, the major dimer has been identified as the cis-syn-uracil dimer (Blackburn and Davies, 1967; Adman et al., 1968; Khattak and Wang, 1968) and the minor one as the cis-anti isomer (Konnert et al., 1970). The combined aqueous extract from 1 g of irradiated uracil was evaporated to dryness. The residue was dissolved in trifluoroacetic acid and chromatographed on paper using solvent system B. Uracil dimer, 6-(4'-(pyrimidin-2 '-one)uracil, and uracil have Rp values 0.10, 0.20, and 0.50, respectively, in this solvent system (Smith, 1963). From the chromatograms, strips (Rw 0.15-0.25) were cut out and extracted with water. The extract was concentrated and chromatographed on a Dowex 50W X-12 (H+) column as described above. Those fractions having an absorbance maximum at 305 nm were combined and evaporated to dryness. The residue, on crystallization from hot water, deposited yellow needle-like crystals of PO-U (Khattak and Wang, 1968) and amounted to 27 mg. Infrared and Ultrauiolet Absorption Spectra. Infrared absorption spectra were recorded on a Perkin-Elmer 337 grating spectrophotometer in potassium bromide pellets. Ultraviolet absorption spectra were recorded using a Hitachi PerkinElmer Coleman 124 double-beam spectrophotometer equipped with a Sargent SRG recorder. Results and Discussion Analysis of paper chromatograms, developed in solvent system A, of cytidine irradiated in frozen aqueous solution, revealed the presence of two products. In the order of chromatographic mobilities, they are referred to as Cdl and Cda. These products were purified by rechromatography in the same solvent. When viewed under an ultraviolet lamp (A 254 nm), Cdl appeared as a narrow dark band (RF 0.09), while cytidine ( R p 0.32) appeared as a broad dark band. When eluted from the chromatograms, Cdl was found to be contaminated with cytidine and Cdz ( R F 0.14). Final purification of Cdl was accomplished by chromatography on the Bio-Gel P-2 column, as described in Methods. On elution from the
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column, Cdn, Cdl, and cytidine appeared in that order. In a typical experiment about 40 mg of CdL was obtained from 3 g of cytidine. The ultraviolet absorption spectrum of CdLin neutral and alkaline solutions shows a poorly resolved peak at 244 nm, which disappears in acidic solution (Figures 1 and 2). Similar spectral characteristics have been previously reported for C&c-saturated cytosine derivatives (Sinsheimer, 1957; Wierzchowski and Shugar, 1962). From a series of absorption spectra taken at pH values from 2 to 8 the pK of CdLwas determined by plotting AClors. pH as 5.4. Though this value is about 1.12 units higher than reported for cytidine (Cavalieri, 1952), it is lower than that reported by Ono et al. (1965) for dihydrocytidine (6.1) and by Johns et al. (1965) for cytidylic acid hydrate (5.56). Irradiation at 254 nm of an aqueous solution of Cdl resulted in an increase in absorbance at 271 nm. Paper chromatogram (solvent A) of the irradiated solution revealed the presence of only one product. On the basis of chromatographic mobilities and ultraviolet absorption spectra in acidic and basic solutions, this product has been identified as cytidine. Thus, the ultraviolet absorption spectrum and photoreversibility indicate that Cd, is probably a cyclobutane-type dimer of cytidine. In aqueous solution, Cdl is converted into different products, depending on the pH of the solution (Scheme I). The conversion proceeds slowly at room temperature and can be hastened by heating. As can be seen from Figure 3, in acidic solution cytidine is the major product, while in alkaline solution uridine is the major detectable product. In aqueous s o h tion (pH 5-8), the main product is cyclobutane-type uridine dimer. Easy deamination of Cs-C6-saturated cytosines to form the corresponding uracils has been reported by other investigators (Green and Cohen, 1957; Daniels and Grimison, 1964). We find that deamination of cyclobutane-type dimeric cytidine is strongly pH dependent, and at pH 2-3 very little deamination takes place, Cdl being converted into cytidine. Cyclobutane-type thymine dimers do not exhibit such monomerization in acidic solution (Herbert et al., 1969). A cyclobutane-type dimer of cytidine could have one of four possible stereoisomeric structures. From both thymidine and uridine irradiated in frozen aqueous solution, three isomeric dimers have been isolated (Fahr, 1968). Our attempts to
PHOTOCHEMICAL REACTIONS OF CYTOSINE NUCLEOSIDES
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PH 3: Conversion of Cd, to different products as a function of pH. (0)Cytidine, ( 0 )uridine dimer, ( X ) uridine. Aqueous solutions of Cdl were heated for 30 min at 100". The solutions were chro-
FIGURE
matographed on paper using solvent system A in which uridine dimer, cytidine, and uridine have RF values 0.16, 0.33, and 0.42, respectively. The amounts of the individual products were determined on the basis of detectable radioactivity.
establish whether Cdl is a mixture of isomeric dimers were unsuccessful. Cyclobutane-Tpe cis-syn-Uracil Dimer from Cdl. AS shown above, Cd, on heating in aqueous solution is converted into different products. These products were separated by Dowex 1X-8 column chromatography with a linear formate gradient. The elution pattern is shown in Figure 4. Fractions 9-14 contained cytidine dimer, 15-18 contained mainly cytidine, 1824 contained cytidine-uridine dimer, probably derived from deamination of one cytosine residue, and fractions 30-45 contained uridine dimer. Prolonged heating of Cdl resulted in lesser amounts of cytidine dimer and cytidine-uridine dimer and a higher amount of uridine dimer. Beyond fractions 70 there was a small peak which, on the basis of photoreversibility, appeared to be a cyclobutane-type uridine dimer. However, the amount was extremely small and further studies were not carried out. A typical experiment in which a solution of Cdl (50 mg/lO ml) was heated for 2 hr in a boiling-water bath, yielded cytidine dimer (5 mg), cytidine (11 mg), cytidine-uridine dimer (4 mg), and uridine dimer (16 mg). The uridine-dimer containing fractions 30-45 were combined, evaporated, desalted, and subjected to mild acid hydrolysis. The hydrolysate was evaporated to dryness and was washed several times with warm methanol. The residue (3.5 mg) was dissolved in hot water and filtered. The filtrate was concentrated to crystallization and left overnight. The ultraviolet and infrared (Figure 6A) absorption spectra of the crystalline product were found to be identical with those of cyclobutane-type uracil cis-syn dimer (V) isolated from uracil. These results suggest that Cdl is mainly cis-syn-cytidine dimer. Characterization of Cd2. When eluted from the chromatograms, Cdl was found to be the major impurity associated with Cds. By rechromatography in the same solvent system, followed by Bio-Gel P-2 column chromatography, pure Cdz was obtained. Cds in aqueous solution exhibited an ultraviolet absorbance maximum at 314 nm. Attempts to crystallize Cd2 were unsuccessful. Cd2 was hydrolyzed with trifluoroacetic acid and chromatographed on paper using solvent system B. From the chromatograms, strips (RF0.12-0.22) were cut out and extracted with
water. The extract was evaporated and subjected to column chromatography in the same manner as that described for the isolation of PO-U from irradiated uracil. Fractions 20-35 having an ultraviolet absorbance maximum at 305 nm were combined and evaporated to dryness. The residue was crystallized from water. The ultraviolet (Figure 5 ) and infrared (Figure 6B) absorption spectra of the crystalline product were found to be identical with those of PO-U (VI) isolated from uracil. Isolation of VI from Irradiated DNA. From the chromatograms, developed in solvent system B, of acid hydrolysates, of irradiated DNA, strips (RF0.05-0.22) were cut out and extracted with water. The combined extract from 3 g of DNA was concentrated and chromatographed on a Dowex 50W X-
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-
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WAVELENGTH ( n m ) FIGURE 5 : Ultraviolet absorption spectra of PO-U isolated from uracil. (A) In water and in 0.1 N HCl; (B) in 0.1 N NaOH. Identical spectra were obtained for PO-U isolated from cytidine and from DNA.
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I
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I300
I200
1100
1000
900
800
700
600
500
FREQUENCY ( C M - ' )
400
FREQUENCY ( CM-' )
6: Infrared absorption spectra of (A) cis-syn-uracil dimer: (B) PO-U isolated from uracil. Identical spectra were obtained for PO-U and uracil dimer isolated from cytidine.
FIGURE
12 column, as described in Methods. Cyclobutane-type uracil dimer and uracil-thymine dimer, which have RF'Sof 0.10 and 0.18, respectively, in solvent B (Smith, 1963), appeared in fractions 3-8. Fractions 20-32, contained a product with ultraviolet absorbance maximum at 305 nm, were combined and evaporated to dryness. The residue was crystallized from water. The ultraviolet (Figure 5 ) and infrared (Figure 6B) absorbance spectra of the product were found to be identical with those of PO-U isolated from uracil. Fractions 3-7, containing the cyclobutane-type dimers, were combined and evaporated to dryness. The residue was dissolved in 0.1 N ammonium hydroxide and chromatographed on a Dowex 1x43 column, as described in Methods. Two peaks were obtained. On the basis of photoreversibility, the first peak was found to be due to uracil-thymine dimer and the second one to uracil dimer. Similar elution patterns were also observed by Weinblum (1967) for cyclobutane-type thymineuracil dimer and uracil-uracil dimer isolated from DNA. Since the amounts were small, further studies to establish the isomeric configurations of these dimers were not made. Irradiation of Thymidine-Cyfidine Mixture. In a previous paper (Varghese, 1970), we showed that thymine dimer, 5thyminyl-5,6-dihydrothymine (IV), and 5-hydroxy-6-(4'pyrimidin-2'-one)dihydrothymine (thymine analog of VII) can be isolated from thymidine irradiated in frozen aqueous solution with ultraviolet light. Irradiation of a mixture of thymidine and cytidine or thymidine and deoxycytidine produce not only the products derived from the individual bases but also products formed from both bases. Thus, following the same procedures as used for the isolation of DNA photo-
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products thymine dimer (Varghese and Wang, 1967a) thymine-uracil dimer, uracil dimer (Weinblum, 1967), and 6-(4'pyrimidin-2'-one)thymine (Varghese and Patrick, 1969) were found to be present in the acid hydrolysates of the irradiated mixture . Mechanism of Formation. Good discussions concerning the mechanism of formation of cyclobutane-type dimers of thymine and uracil derivatives have appeared in the literature (Eisinger and Shulman, 1968; Lamola and Eisinger, 1968). For thymine it has been demonstrated that photodimerization can proceed by way of a triplet-state precursor in dilute aqueous solution, while a singlet is directly involved in dimerization in frozen aqueous solution. Since cyclobutane-type dimer formation of cytidine can be photosensitized by acetone in a manner analogous to that of thymidine (A. J. Varghese, manuscript in preparation), it is reasonable to assume that photodimerization of cytosine nucleosides and thymidine occurs by the same mechanism. The structure of the dehydration product of a uracil-uracil adduct was established as 6-(4'-(pyrimidin-2 '-one)uracil (VI) (Khattak and Wang, 1968). We have isolated the same product from uridine irradiated in frozen aqueous solution with ultraviolet light (A. J. Varghese, manuscript in preparation). The mechanism of formation of uracil-uracil adduct VI11 probably involves an oxetane intermediate VI1 similar to the one proposed for the formation of a thymine-thymine adduct (Varghese and Wang, 1968). Even though the exact structure of Cd? is not known, the formation of PO-U from Cd2 by acid hydrolysis suggests that Cd2 may be a cytidine-cytidine adduct X. The formation of
P H O T O C H E M I C A L R E A C T I O N S OF C Y T O S I N E NUCLEOSIDES
Acknowledgments
0
0
The author is indebted to Drs. D. Creed, J. Jagger, M. H. Patrick, and C. S . Rupert for helpful discussions. The valuable technical assistance of Mrs. Mary-Pat Hogan and Miss Suzanna Yung is gratefully acknowledged.
B
I
R
VIII
VII
X can be explained by an initially formed azetidine intermediate IX. Such an azetidine intermediate, formed from cytosine and thymine residues in DNA, has been proposed to explain the formation of 6-(4'-pyrimidin-2'-one)thymine in irradiated DNA (Wang and Varghese, 1967). Thus, VI constitutes the second such product isolated from irradiated DNA. The
n
"2
IX
X
possible biological significanceof this class of products, having characteristic absorption maxima above 300 nm, is not yet clear. Jagger et al. (1970) have shown that an unusual kind of photoreactivation, which these authors refer to as type 111 photoreactivation, occurs at 3 13 nm in Streptomyces griseus and Streptomyces coelicolor, inactivated with ultraviolet light. A noteworthy feature of these organisms is the high guaninecytosine content of its DNA (74% G C, Belozersky and Spirin, 1960). It is possible that photoproducts related to POU might play a significant role in the photoreactivation of these organisms. In TMV RNA, Small et al. (1968) have shown that irradiation at high ionic strength results in the formation of two dimeric products, referred to as photoproducts I and 11, which are not cyclobutane-type dimeric uracils. These investigators have shown that these unidentified products and two cyclobutane-type dimeric products are responsible for the inactivation by ultraviolet light of TMV RNA at high ionic strength. It is possible that photoproducts I and I1 are derivatives or precursors of PO-U. The procedure for determining the amount of cyclobutanetype dimers of cytosine in DNA is based on the amount of detectable urdcil dimer in acid hydrolysates of irradiated DNA. n h e procedure also involves heating the irradiated DNA prior to hydrolysis to convert the cytosine dimers to uracil dimers (Setlow et al., 1965).] Our results show that cytidine dimer is not converted completely into uracil dimer under any condition. Moreover, acid hydrolysis of cis-synuridine dimer produces a mixture of uracil and cis-syn-uracil dimer. Therefore, it is possible that the contribution of cyclobutane-type cytosine dimers in the inactivation of biological systems might be much greater than could be accounted for by the detectable uracil dimer in acid hydrolysates of irradiated DNA.
+
References Adman, E., Gordon, M. P., and Jessen, L. H. (1968), Chem. Commun., 1019. Belozersky, A. J., and Spirin, A. S. (1960), Nucl. Acids 3,147. Blackburn, G. M., and Davies, R. J. H. (1967), Tetrahedron Lett. 37,4471. Burr, J. G . (1968))Aduan. Photochem. 6,193. Cavalieri, L. (1952), J. Amer. Chem. SOC.74,5804. Daniels, M., and Grimison, A. (1964))Biochem. Biophys. Res. Commun. 16,428. Eisinger, J., and Shulman, R. G . (1968), Science 161,1311. Fahr, E. (1968), Angew. Chem., Engl. Znt. Ed. 8,578. Green, M., and Cohen, S. S. (1957), J . Biol. Chem. 228,601. Haug, A. (1964), Photochem. Photobiol. 3,207. Herbert, M. A., LeBlanc, J. C., Weinblum, D., and Johns, H. E. (1969), Photochem. Photobiol. 9,33. Jagger, J., Takebe, H., and Snow, J. M. (1970), Photochem. Photobiol. 12,185. Johns, H. E., LeBlanc, J. C., and Freeman, K. B. (1965), J . Mol. Biol. 13,849. Khattak, M. N., and Wang, S. Y. (1968), Science 163, 1341. Konnert, J., Gibson, J. W., Karle, I. L., Khattak, M. N., and Wang, S. Y .(1970), Nature (London)227,953. Lamola, A. A., and Eisinger, J. (1968), Proc. Nut. Acad. Sci. U . S. 59,46. McLaren, A. D., and Shugar, D. (1964)) Photochemistry of Proteins and Nucleic Acids, New York, N. Y . , Macmillian, p 174. Miller, N., and Cerutti, P. (1968), Proc. Nut. Acad. Sci. U. S. 59,34. Ono, J. Wilson, R. G., and Grossman, L. (1965), J . Mol. Biol. 11, 600. Setlaw, R. B., and Carrier, W. L. (1963), Photochem. Photobiol. 2,49. Setlow, R. B., and Carrier, W. L. (1966), J. Mol. Biol. 17, 237. Setlow, R. B., Carrier, W. L., and Bollum, F. J. (1965), Proc. Nut. Acad. Sci. U . S. 53,1111. Sinsheimer, R. L. (1957), Radial. Res. 6,121. Small, D. G., Tao, M., and Gordon, M. P. (1968), J. Mol. Biol. 3475. Smith, K . C.(1963))Photochem. Photobiol. 2, 503. Varghese, A. J. (1970))Biochemistry 9,4781. Varghese, A. J., and Patrick, M. H. (1969), Nature (London) 223,299. Varghese, A. J., and Rupert, C. S. (1971), Photochem. Photobiol. 13,365. Varghese, A. J., and Wang, S. Y . (1967a), Nature (London) 213,909. Varghese, A. J., and Wang, S. Y. (1967b))Science 156,955. Varghese, A. J., and Wang, S. Y .(1968),Science 160,186. Wang, S. Y . , and Varghese, A. J. (1967), Biochem. Biophys. Res. Commun. 29,543. Weinblum, D. (1967), Biochem. Biophys. Res. Commun. 27,384. Weinblum, D., and Johns, H. E. (1965), Biochim. Biophys. Acta 114,450. Wierzchowski, K. L., and Shugar, D. (1962), Photochem. Photobiol. 1,21. B I O C H E M I S T R Y , VOL.
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